A fast, cost-effective, and highly sensitive method was developed for the determination of five barbiturates with four internal
standards (ISTDs) in urine using an Agilent 6430 Triple Quadrupole LC–MS system and an Agilent Poroshell 120 EC-C18 column.
The sample of urine was extracted using an Agilent SPEC-C18AR cartridge. Results indicate that the method effectively extracts
the selected barbiturates from urine, resolving the target compounds and the ISTDs in 8.5 min. Though amobarbital and pentobarbital
differ only in the position of a methyl group and are, therefore, difficult to separate, their resolution is sufficient for
Materials and Methods
All compounds were purchased from Cerilliant Corporation, Round Rock, Texas, United States.
1. Begin by centrifuging the urine sample at 2800 rpm for 5 min.
2. Pipette 1 mL of centrifuged sample into a 13 mm × 100 mm borosilicate glass tube.
3. Add exactly 35 µL of the working deuterated internal standard (butalbital-D5, pentobarbital-D5, secobarbital-D5, and phenobarbital-D5).
4. Pipette 500 µL 0.1 M phosphate buffer into the sample. The phosphate buffer is prepared by adding 13.61 g KH2PO4 into 800 mL water, adjusting to pH 6.0 with KOH, then making the volume up to 1 L.
5. Use a vacuum chamber with the Agilent SPEC-C18AR cartridge for extraction. Condition the cartridge with 0.2 mL of MeOH
and load the sample solution.
6. Wash the column with 0.5 mL water and dry for 1 min.
7. Elute the cartridge with 1 mL 90:10 hexane:ethyl acetate mixture.
8. Collect the eluent and dry the sample under nitrogen gas at 35 °C.
9. Reconstitute with 0.5 mL 90:10 water:acetonitrile mixture.
The method was performed on the Agilent 1260 Infinity LC with a 6430 Triple Quadrupole LC–MS.
Column: Agilent Poroshell 120 EC-C18, 2.1 mm × 100 mm, 2.7 µm (p/n 695775-902)
Sample preparation: Agilent SPEC-C18CR, 3 mL, 15 mg (p/n A5321920); Eluent A, 5 mM ammonium acetate; Eluent B, LC–MS grade acetonitrile
ESI drying gas: 350 °C, 10 L/min
Figure 1: MRM chromatograms of barbiturates and internal standards using an Agilent Poroshell 120 EC-C18 column.
Nebulizer: 40 psi
Negative ionization mode
Capillary: 4000 V, DEMV 400
Note: If using an instrument that is not configured for low delay volume, use about 1 minute initial hold.